1. The cancer stem cell model posits that the parenchymal cells of tumor are hierarchically organized and at the top of this hierarchy are cells that are uniquely capable of initiating and sustaining tumorigenesis. These cancer stem cells have the ability to self-renew creating a more diverse and differentiated population of cells that are nontumorigenic and make up the bulk of the tumor while maintaining a population of cancer stem cells through the process of self-renewal. Understanding the properties of cancer stem cells, including enhanced therapeutic resistance resulting from high expression of multidrug transporters, enhanced DNA damage checkpoint activation and repair mechanisms, and altered cell-cycle kinetics is critical to the development of more effective cancer therapies. Towards the goal of identifying and tracking the behavior of cancer stem cells in vivo, our collaborators have developed a functional imaging approach to cancer stem cell identification using the central triad of master transcriptional regulators, OCT4, SOX2, and NANOG to drive a fluorescent reporter construct that would uniquely identify cancer stem cells. In this study, we have capitalized on our expertise in time-lapse optical microscopy to follow cancer stem cell migration in the tumor environment validating our ability to identify and track the movement of cells through their expression of a fluorescent protein construct whose cellular production requires the transcriptional regulator triad associated with cancer stem cells. 2. Since 2001, over 150,000 US military personnel have been diagnosed with mild traumatic brain injury or concussion, often after exposure to an explosive blast, with a spectrum of neurological and psychological deficits. Understanding the mechanisms and pathology resulting from the primary injury phase of a blast, a direct result of the shockwave generated by an explosion, is still quite limited. The brain is a complex system with compositional inhomogeneity through which shock waves travel at different speeds; these differences in speed have the potential to create shearing forces between and within brain cells. We previously developed an advance system that mimics the temporal properties of a blast shock wave and is integrated with a state-of-the-art optical microscope allowing us to observed directly the effect of a blast on brain cells. This system allowed us to reproducibly deliver blast shock waves with and without shear forces to the same cells at different points of time and to follow their responses for 24 hours. We demonstrated that brain cells in culture are indifferent to blast induced transient pressure waves known to cause mild blast induced traumatic brain injury. However, when sufficient shear forces are present with shockwave pressure, calcium waves propagate throughout the cellular network of central nervous system dissociated cultures. These results suggested that shear forces have a role in how blast shock wave exposure leads to mild traumatic brain injury and call attention to the need to characterize the response of brain cells to shear in the absence of pressure transients. Previous work indicated that the rise time of an insult shear force is an important parameter; faster rise times have larger cellular effects. However, no device existed that was capable of delivering shear forces on the time scale of an explosive blast. Toward the goal of better understanding traumatic brain injury, in particular, the primary injury phase associated with mild blast induced traumatic brain injury, we developed and were awarded a provisional patent for a new system that allows precise control of shear forces over a time interval associated with a blast shock wave: sub msec rise times and msec durations. Our system is based on a microfluidic platform that is compatible with high resolution, time-lapse, optical microscopy. We established that the parameters of the flow field during a blast agreed with theory. The growth and survival of dissociated brain cell cultures within the microfluidic chamber system was verified. This new system has allowed us to examine the effects of shear forces on brain cells in culture under conditions that mimic the temporal properties of a blast shock wave without the accompanying pressure transients, effectively decoupling the two forces, pressure and shear, that are critical to the pathophysiology associated with traumatic brain injury. 3. We have continued our long-term study of membrane protein domains, using the textbook example of influenza hemagglutinin domains on the plasma membrane of fibroblasts. The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.